Radiotherapy is a useful component of treatment strategies for esophageal cancer. The role of autophagy in response to ionizing radiation was investigated in human esophageal squamous carcinoma cells. Cell viability and clonogenic survival assay were used to evaluate the radiosensitivity of autophagy inhibitor (3-MA) on esophageal squamous carcinoma cells. The percentage of apoptotic cells and cell cycle analysis were assessed by flow cytometry; DAPI staining was used to detect apoptotic cells. The expression of beclin-1 and LC3 was measured using a Western blot. The ultrastructural analysis was under the electron microscope. 6 Gy irradiation induced a massive accumulation of autophagosomes accompanied by strong upregulation of beclin-1 and LC3-II expression in TE-1 cells. Compared with radiation alone, 3-MA combined with radiation significantly decreased cell viability, as well as autophagic ratio, beclin-1, and LC3-II protein level. Inhibition of autophagy increased radiation-induced apoptosis and the percentage of G2/M-phase cells. Blockade of autophagy with 3-MA enhanced cytotoxicity of radiotherapy in human esophageal squamous carcinoma cells. It suggests that inhibition of autophagy could be used as adjuvant therapy to treat esophageal squamous cell carcinoma.